DESCRIPTION: (taken from the application). The precise molecular targets of general anesthetics and their binding sites are unknown and the relative contribution of presynaptic versus postsynaptic effects for both the desired and undesired effects of general anesthetics are poorly understood. This proposed study will examine the quantitative impact of representative general anesthetics on parameters controlling presynaptic physiology and on molecular interactions of key presynaptic molecular components. The overall objectives are to determine the subcellular processes, and their molecular substrates, that are targets for general anesthetic action in the central nervous system (CNS) by combining physiological and biochemical approaches to this problem. The long-term goal of the proposed studies is to define the effects of general anesthetics on presynaptic function in order to target the rational development of more specific anesthetics with potentially fewer adverse effects. The physiological approach uses the fluorescent tracer FM 1-43 and new genetically encoded fluorescent reporters or Ca2+ sensitive fluorescent dyes to study anesthetic effects on synaptic vesicle exocytosis, endocytosis (recycling) and Ca2+ entry in single presynaptic terminals of hippocampal cell cultures. These experiments will provide detailed information of anesthetic actions on the physiological parameters that control synaptic efficiency at the single synapse level. The biochemical approach is to study anesthetic effects on neurotransmitter secretion in biochemically accessible permeabilized synaptosomes and on protein interactions between identified components of the fusion/exocytosis machinery in brain extracts or using purified recombinant proteins. Our working hypothesis is that general anesthetics alter neurotransmitter release by agent- and transmitter-specific effects on identifiable steps in the synaptic vesicle exocytosis/endocytosis cycle.